Identification and manipulation of tumor associated macrophages in
600 200 800 200 means for controlling tumor associated ...€¦ · means for controlling tumor...
1
APL-102, an oral small molecule multi-kinase inhibitor, demonstrates favorable CSF-1R activity, offering a means for controlling tumor associated macrophages Elaine Liu 1 , Lan Yang 1 , Gavin Choy 2 , Xiaoling Zhang 2 , Tillman Pearce 2 , Mamatha Reddy 2 , Sanjeev Redkar 2 , Qian Shi 1 1 Zhejiang Apollomics Biotech Co., Ltd., Hangzhou, China, 2 Apollomics, Inc., Foster City, CA, USA Background APL-102 (previously CBT-102) is a multi-kinase inhibitor with a unique inhibitory profile. Previous presentation (Abstract #3945, AACR 2018) showed potent activity of APL-102 on VEGFR, BRAF, CRAF, key kinases in angiogenesis, MAPK pathways, and demonstrated in vivo efficacies in a broad spectrum of PDX models. The role of the tumor microenvironment (TME) in fostering the development of malignancies is prompting the pursuit of anticancer therapies that target its components as opposed to the tumor itself. As part of their immune surveillance duties, immune cells form part of this microenvironment, and yet, cancer cells have devised means to downplay their tumoricidal capabilities. Colony stimulating factor 1 receptor (CSF-1R) may offer such a means of controlling tumor associated macrophages in the tumor microenvironment. Materials and Methods The potency and selectivity of APL-102 against CSF1R kinase was evaluated in a cell free system where the enzyme activity was evaluated with Eurofins standard KinaseProfiler TM system. Cell growth inhibition was evaluated in the Ba/F3 hCSF1R cell line by the CellTiter- Glo (CTG) method. Engineered cell line Ba/F3 hCSF1R was treated with APL-102 and control compounds for 3 days at 37°C, 5% CO 2 and 95% humidity. The effect of APL-102 was further evaluated in murine myelogenous cell lines and human monocytes. Two murine cell lines M-NFS-60 and RAW264.7 were starved and then treated with APL-102 or comparator test articles for 3 days at 37°C, 5% CO 2 and 95% humidity. Human monocytes was isolated from PBMC and treated with APL-102 or control compounds for 5 days at 37°C, 5% CO 2 and 95% humidity. In vivo efficacy studies of APL-102 in combination with an anti-PD-1 antibody were performed in two immunotherapy sensitive syngeneic models, a subcutaneous MC-38 colon model in C57BL/6J mice and a CT-26 colon model in BALB/c mice. Treatment with APL-102 and anti- PD-1 antibody as single agent and in combination were initiated when tumors reached a mean volume of 100－140 mm 3 . Tumor volumes were measured twice weekly in two dimensions using a caliper, and the volume was calculated using the formula: V = (L × W × W)/2. Tumor growth inhibition rate (TGI%) was used to evaluate the efficacy of in vivo studies. Results AACR Annual Meeting 2019, March 29 – April 3, 2019, Atlanta, Georgia, USA Abstract No.: #2205 Synergistic effect of APL-102 combined with anti-PD-1 Figure 3: APL-102 demonstrated selective inhibition of the CSF-1 dependent M-NFS-60 cell line compared to CSF-1 independent RAW264.7 cell and human monocytes. In contrast, the three CSF1R targeting benchmark compounds GW2580, BLZ945, and PLX3397 showed highest inhibition of human monocyte. The results demonstrated that APL-102 inhibits CSF1R in a radiometric enzyme activity assay with an IC50 of 43 nM. APL-102 demonstrated growth inhibition in cells dependent on CSF1-CSF1R signaling. We demonstrated a mechanism of action for APL-102 anti-tumor activity, the inhibition of CSF1R-dependent tumor associated macrophages in TME in addition to targeting VEGFR-dependent angiogenesis and MAPK pathway. Combination of APL-102 and anti-PD-1 produced a more robust response than either single agent alone in syngeneic mouse models, which was associated with macrophage inhibition in TME. Rational combination with check-point inhibitors (CPIs) may improve the efficacy of APL-102 and broaden the efficacy of CPIs. Further studies may be needed to delineate the interplay among APL-102’s different mechanisms and its synergistic effects in combination with CPIs. Contact / Further Information Please visit Apollomics website at www.apollomicsinc.com for a PDF version of the poster presentation. APL-102 CSF1R Inhibition Cell line APL-102 GW2580 BLZ945 PLX3397 IC50 (μM) Max inh.(%) IC50 (μM) Max inh.(%) IC50 (μM) Max inh.(%) IC50 (μM) Max inh.(%) M-NFS-60 0.631 99.44% 1.145 99.02% 3.015 99.08% 0.348 99.32% RAW264.7 22.85 95.44% 4.056 81.91% >30 24.65% 16.36 95.16% Monocyte 6.98 96.05% 0.28 96.57% 0.20 96.34% 0.06 97.99% Figure 1: APL-102 inhibition of CSF1R enzyme activity was dose-dependent, with IC50 of 43 nM. The result was obtained with a radiometric enzymatic activity assay. Figure 2: In engineered Ba/F3 hCSF1R cell lines, the IC50 value of APL-102 was 0.588 μM compared to 1.333 μM of sulfatinib, a similar multi-kinase inhibitor, and 0.279 μM of GW2580, a more specific CSF1R kinase inhibitor. IC50 determination on M-NFS-60 0.001 0.01 0.1 1 10 100 0 50 100 150 200 200 400 600 800 CBT-102 GW2580 BLZ945 PLX3397 Conc. uM Surviving cells(%) IC50 determination on Monocyte 0.001 0.01 0.1 1 10 100 0 20 40 60 80 100 120 CBT-102 GW2580 BLZ945 PLX3397 Conc. uM Surviving cells(%) Summary IC50=43 nM Group Treatment Dose (mg/kg) TV (mm 3 ) Day 21 TGI (%) T/C (%) P value 1 Vehicle 0 1974.55±239.30 -- -- -- 2 APL-102 *10(5) 957.58±164.28 51.50 48.50 0.032 3 Anti-PD-1 antibody 10 1239.28±571.38 37.24 62.76 0.329 4 APL-102 *10(5) 479.07±144.00 75.74 24.26 0.001 Anti-PD-1 antibody 10 Figure 4: The efficacy study of APL-102 combined with an anti-PD-1 antibody in CT-26 colon cancer model. The results demonstrated effect in the combination of APL-102 with anti-PD-1 antibody. The TGI% was 75.74% in combination group, which was stronger than the single agent groups respectively. * : Groups 2 and 4 had changed doses from 10 to 5mpk during the experiment. Group Treatment Dose (mg/kg) TV (mm 3 ) Day 28 TGI (%) T/C (%) P value 1 Vehicle 0 2668.22±353.34 -- -- -- 2 APL-102 5 768.38±45.83 72.20 28.80 <0.001 3 APL-102 10 408.88±62.85 84.68 15.32 <0.001 4 Anti-PD-1 antibody 10 1180.07±190.60 55.77 44.23 0.003 5 APL-102 5 212.23±41.79 92.05 7.95 <0.001 Anti-PD-1 antibody 10 6 APL-102 10 263.34±42.43 90.13 9.87 <0.001 Anti-PD-1 antibody 10 Figure 5: Efficacy study of APL-102 combined with anti-PD-1 antibody in MC-38 colon cancer model. APL-102 showed significant inhibition on MC-38 tumor growth as a single agent. Anti-PD-1 antibody also showed inhibition as a single agent. The combination of APL-102 with anti-PD-1 antibody showed improved activity. Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 A. CD3 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 B. CD4 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 C. CD8 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 D. F4/80 Figure 6: Expression of CD3, CD4, CD8, and F4/80 markers in tumor tissues tested by IHC in the MC-38 efficacy study. Group 1 was treated with vehicle, Group 2 was treated with 5 mg/kg of APL-102, Group 3 was treated with 10 mg/kg of APL-102, Group 4 was treated with 10 mg/kg of anti-PD-1 antibody, Group 5 was combined treatment with 5 mg/kg APL-102 and 10 mg/kg anti-PD-1 antibody, Group 6 was combined treatment with 10 mg/kg APL-102 and 10 mg/kg anti-PD-1 antibody. The results demonstrated that APL-102 increased the total T cells (CD3, Panel A), CD8 T cells (CD8, Panel C), but had minimal effect on CD4 T cells (CD4, Panel B). At the same time, APL-102 treatment significantly decreased macrophages in the tumor, both as single agent and in combination with an anti-PD-1 antibody. Vehicle APL-102 5 mpk APL-102 10 mpk aPD1 10 mpk APL-102+aPD1 5 mpk +10 mpk APL-102+aPD1 10 mpk+10 mpk APL-102 0 500 1000 1500 2000 2500 3000 3500 10 13 16 19 22 25 28 Tumor Volume (mm³) Treatment Days Efficacy study of APL-102 combined anti-PD-1 antibody in MC-38 colon cancer model Group 01, Vehicle, 0 mg/kg, QDx21days, p.o. Group 02, APL-102, 5 mg/kg, QDx21days, p.o. Group 03, APL-102, 10 mg/kg, QDx21days, p.o. Group 04, Anti-PD-1, 10 mg/kg, BIWx3weeks, i.p. Group 05, APL-102, 5 mg/kg, QDx21days, p.o., Anti-PD-1, 10 mg/kg, BIWx3weeks, i.p. Group 06, APL-102, 10 mg/kg, QDx21days, p.o., Anti-PD-1, 10 mg/kg, BIWx3weeks, i.p. APL-102 APL-102 IC50 determination on RAW264.7 0.001 0.01 0.1 1 10 100 0 50 100 150 200 250 CBT-102 GW2580 BLZ945 PLX3397 Conc. uM Surviving cells(%) APL-102 0 500 1000 1500 2000 2500 6 9 12 15 18 21 Tumor Volume (mm³) Treatment Days Efficacy study of APL-102 combined anti-PD-1 antibody in CT-26 colon cancer model Group 01, Vehicle, 0 mg/kg, QD*15days p.o. Group 02, APL-102, adjust 10 mg/kg, QD*6days, p.o. to 5 mg/kg, QD*9days since Day13 Group 03, anti-PD-1, 10 mg/kg, BIW*5times i.p. Group 04, APL-102, adjust 10 mg/kg, QD*6days, p.o. to 5 mg/kg, QD*9days since Day13； anti-PD-1, 10 mg/kg, BIW*5times i.p.
Transcript of 600 200 800 200 means for controlling tumor associated ...€¦ · means for controlling tumor...
APL-102, an oral small molecule multi-kinase inhibitor, demonstrates favorable CSF-1R activity, offering a
means for controlling tumor associated macrophagesElaine Liu1, Lan Yang1, Gavin Choy2, Xiaoling Zhang2, Tillman Pearce2, Mamatha Reddy2, Sanjeev Redkar2, Qian Shi1
1Zhejiang Apollomics Biotech Co., Ltd., Hangzhou, China, 2Apollomics, Inc., Foster City, CA, USA
Background
APL-102 (previously CBT-102) is a multi-kinase inhibitor with a unique inhibitory profile.
Previous presentation (Abstract #3945, AACR 2018) showed potent activity of APL-102 on
VEGFR, BRAF, CRAF, key kinases in angiogenesis, MAPK pathways, and demonstrated in
vivo efficacies in a broad spectrum of PDX models.
The role of the tumor microenvironment (TME) in fostering the development of malignancies is
prompting the pursuit of anticancer therapies that target its components as opposed to the tumor
itself. As part of their immune surveillance duties, immune cells form part of this
microenvironment, and yet, cancer cells have devised means to downplay their tumoricidal
capabilities. Colony stimulating factor 1 receptor (CSF-1R) may offer such a means of
controlling tumor associated macrophages in the tumor microenvironment.
Materials and Methods
The potency and selectivity of APL-102 against CSF1R kinase was evaluated in a cell free
system where the enzyme activity was evaluated with Eurofins standard KinaseProfilerTM
system. Cell growth inhibition was evaluated in the Ba/F3 hCSF1R cell line by the CellTiter-
Glo (CTG) method. Engineered cell line Ba/F3 hCSF1R was treated with APL-102 and control
compounds for 3 days at 37°C, 5% CO2 and 95% humidity.
The effect of APL-102 was further evaluated in murine myelogenous cell lines and human
monocytes. Two murine cell lines M-NFS-60 and RAW264.7 were starved and then treated with
APL-102 or comparator test articles for 3 days at 37°C, 5% CO2 and 95% humidity. Human
monocytes was isolated from PBMC and treated with APL-102 or control compounds for 5 days
at 37°C, 5% CO2 and 95% humidity.
In vivo efficacy studies of APL-102 in combination with an anti-PD-1 antibody were performed
in two immunotherapy sensitive syngeneic models, a subcutaneous MC-38 colon model in
C57BL/6J mice and a CT-26 colon model in BALB/c mice. Treatment with APL-102 and anti-
PD-1 antibody as single agent and in combination were initiated when tumors reached a mean
volume of 100－140 mm3. Tumor volumes were measured twice weekly in two dimensions
using a caliper, and the volume was calculated using the formula: V = (L × W × W)/2. Tumor
growth inhibition rate (TGI%) was used to evaluate the efficacy of in vivo studies.
Results
AACR Annual Meeting 2019, March 29 –April 3, 2019, Atlanta, Georgia, USA
Abstract No.: #2205
Synergistic effect of APL-102 combined with anti-PD-1
Figure 3: APL-102 demonstrated selective inhibition of the CSF-1 dependent M-NFS-60 cell line compared
to CSF-1 independent RAW264.7 cell and human monocytes. In contrast, the three CSF1R targeting
benchmark compounds GW2580, BLZ945, and PLX3397 showed highest inhibition of human monocyte.
The results demonstrated that APL-102 inhibits CSF1R in a radiometric enzyme activity assay with an IC50 of
43 nM. APL-102 demonstrated growth inhibition in cells dependent on CSF1-CSF1R signaling.
We demonstrated a mechanism of action for APL-102 anti-tumor activity, the inhibition of CSF1R-dependent
tumor associated macrophages in TME in addition to targeting VEGFR-dependent angiogenesis and MAPK
pathway. Combination of APL-102 and anti-PD-1 produced a more robust response than either single agent
alone in syngeneic mouse models, which was associated with macrophage inhibition in TME. Rational
combination with check-point inhibitors (CPIs) may improve the efficacy of APL-102 and broaden the efficacy
of CPIs. Further studies may be needed to delineate the interplay among APL-102’s different mechanisms and
its synergistic effects in combination with CPIs.
Contact / Further Information
Please visit Apollomics website at www.apollomicsinc.com for
a PDF version of the poster presentation.
APL-102 CSF1R Inhibition
Cell line
APL-102 GW2580 BLZ945 PLX3397
IC50 (μM) Max inh.(%) IC50 (μM) Max inh.(%) IC50 (μM) Max inh.(%) IC50 (μM) Max inh.(%)
M-NFS-60 0.631 99.44% 1.145 99.02% 3.015 99.08% 0.348 99.32%
RAW264.7 22.85 95.44% 4.056 81.91% >30 24.65% 16.36 95.16%
Monocyte 6.98 96.05% 0.28 96.57% 0.20 96.34% 0.06 97.99%
Figure 1: APL-102 inhibition of CSF1R
enzyme activity was dose-dependent, with
IC50 of 43 nM. The result was obtained with
a radiometric enzymatic activity assay.
Figure 2: In engineered Ba/F3 hCSF1R cell lines,
the IC50 value of APL-102 was 0.588 µM
compared to 1.333 µM of sulfatinib, a similar
multi-kinase inhibitor, and 0.279 µM of GW2580,
a more specific CSF1R kinase inhibitor.
IC50 determination on M-NFS-60
0.001 0.01 0.1 1 10 1000
50
100
150
200200
400
600
800CBT-102
GW2580
BLZ945
PLX3397
Conc. uM
Su
rviv
ing
cells(%
)
IC50 determination on Monocyte
0.001 0.01 0.1 1 10 1000
20
40
60
80
100
120CBT-102
GW2580
BLZ945
PLX3397
Conc. uM
Su
rviv
ing
cells(%
)
Summary
IC50=43 nM
Group TreatmentDose
(mg/kg)
TV (mm3)
Day 21TGI (%) T/C (%) P value
1 Vehicle 0 1974.55±239.30 -- -- --
2 APL-102 *10(5) 957.58±164.28 51.50 48.50 0.032
3Anti-PD-1
antibody10 1239.28±571.38 37.24 62.76 0.329
4
APL-102 *10(5)
479.07±144.00 75.74 24.26 0.001Anti-PD-1
antibody10
Figure 4: The efficacy study of APL-102 combined with an anti-PD-1 antibody in CT-26 colon cancer model.
The results demonstrated effect in the combination of APL-102 with anti-PD-1 antibody. The TGI% was
75.74% in combination group, which was stronger than the single agent groups respectively.
* : Groups 2 and 4 had changed doses from 10 to 5mpk during the experiment.
Group TreatmentDose
(mg/kg)
TV (mm3)
Day 28TGI (%) T/C (%) P value
1 Vehicle 0 2668.22±353.34 -- -- --
2 APL-102 5 768.38±45.83 72.20 28.80 <0.001
3 APL-102 10 408.88±62.85 84.68 15.32 <0.001
4Anti-PD-1
antibody10 1180.07±190.60 55.77 44.23 0.003
5
APL-102 5
212.23±41.79 92.05 7.95 <0.001Anti-PD-1
antibody10
6
APL-102 10
263.34±42.43 90.13 9.87 <0.001Anti-PD-1
antibody10
Figure 5: Efficacy study of APL-102 combined with anti-PD-1 antibody in MC-38 colon cancer model.
APL-102 showed significant inhibition on MC-38 tumor growth as a single agent. Anti-PD-1 antibody also
showed inhibition as a single agent. The combination of APL-102 with anti-PD-1 antibody showed improved
activity.
Group 1 Group 2 Group 3 Group 4 Group 5 Group 6A.
CD3
Group 1 Group 2 Group 3 Group 4 Group 5 Group 6B.
CD4
Group 1 Group 2 Group 3 Group 4 Group 5 Group 6C.
CD8
Group 1 Group 2 Group 3 Group 4 Group 5 Group 6D.
F4/80
Figure 6: Expression of CD3, CD4, CD8, and F4/80 markers in tumor tissues tested by IHC in the MC-38
efficacy study. Group 1 was treated with vehicle, Group 2 was treated with 5 mg/kg of APL-102, Group 3 was
treated with 10 mg/kg of APL-102, Group 4 was treated with 10 mg/kg of anti-PD-1 antibody, Group 5 was
combined treatment with 5 mg/kg APL-102 and 10 mg/kg anti-PD-1 antibody, Group 6 was combined
treatment with 10 mg/kg APL-102 and 10 mg/kg anti-PD-1 antibody. The results demonstrated that APL-102
increased the total T cells (CD3, Panel A), CD8 T cells (CD8, Panel C), but had minimal effect on CD4 T
cells (CD4, Panel B). At the same time, APL-102 treatment significantly decreased macrophages in the tumor,
both as single agent and in combination with an anti-PD-1 antibody.
VehicleAPL-102
5 mpk
APL-102
10 mpk
aPD1
10 mpk
APL-102+aPD1
5 mpk +10 mpk
APL-102+aPD1
10 mpk+10 mpk
APL-102
0
500
1000
1500
2000
2500
3000
3500
10 13 16 19 22 25 28
Tum
or
Vo
lum
e (
mm
³)
Treatment Days
Efficacy study of APL-102 combined anti-PD-1 antibody in
MC-38 colon cancer model
Group 01, Vehicle, 0 mg/kg, QDx21days, p.o.
Group 02, APL-102, 5 mg/kg, QDx21days, p.o.
Group 03, APL-102, 10 mg/kg, QDx21days, p.o.
Group 04, Anti-PD-1, 10 mg/kg, BIWx3weeks, i.p.
Group 05, APL-102, 5 mg/kg, QDx21days, p.o., Anti-PD-1,
10 mg/kg, BIWx3weeks, i.p.
Group 06, APL-102, 10 mg/kg, QDx21days, p.o., Anti-PD-1,
10 mg/kg, BIWx3weeks, i.p.
APL-102 APL-102
IC50 determination on RAW264.7
0.001 0.01 0.1 1 10 1000
50
100
150
200
250CBT-102
GW2580
BLZ945
PLX3397
Conc. uM
Su
rviv
ing
cells(%
)
APL-102
0
500
1000
1500
2000
2500
6 9 12 15 18 21
Tum
or
Vo
lum
e (
mm
³)
Treatment Days
Efficacy study of APL-102 combined anti-PD-1 antibody in
CT-26 colon cancer model
Group 01, Vehicle, 0 mg/kg, QD*15days p.o.
Group 02, APL-102, adjust 10 mg/kg, QD*6days, p.o. to 5 mg/kg,
QD*9days since Day13Group 03, anti-PD-1, 10 mg/kg, BIW*5times i.p.
Group 04, APL-102, adjust 10 mg/kg, QD*6days, p.o. to 5 mg/kg,
QD*9days since Day13； anti-PD-1, 10 mg/kg, BIW*5times i.p.
